高分子表面微图案技术研究干细胞形状、尺寸和密度对其分化的影响

发布时间：2018-11-08 08:51

【摘要】：细胞与材料相互作用是生物材料学、细胞生物学和再生医学的一个基本科学问题,相关研究对于生物材料的设计具有重要的指导意义。细胞作为生命活动的基本单元,对单细胞的研究能直接反映细胞内本质的变化。但由于高效的单细胞研究手段的相对不足,这方面的进展仍然不够。微米图案技术,能在基底上实现细胞黏附反差,从而能控制细胞的定位,经调节细胞可黏附区域即黏附岛的大小可以有效地实现单细胞的黏附,且样本数量大,可用于统计而得到可信的结论,在对单细胞的研究中有很好的应用前景。组织器官不是由单细胞组成的,而是多个或多种细胞及胞外的基质等组成的一个大社会。细胞与细胞之间相互作用也是组织工程中重要的基础科学问题。多细胞的聚集程度,即细胞密度也非常重要。微米图案技术,通过控制细胞的定位,从而可以有效地控制细胞与细胞的接触、细胞的聚集程度等,是研究多细胞之间相互作用的有效手段。骨髓基质干细胞具有多向分化潜能且易于体外扩增,取自成体而不涉及伦理范畴,是组织工程理想的种子细胞类型。干细胞分化是当今白然科学的研究前沿。本博士论文选用骨髓基质干细胞为模型,借助高分子基底上独到的图案化技术来研究影响干细胞分化的各种物理因素,包括单细胞分化的形状效应、尺寸效应,以及多细胞状态下干细胞分化的密度效应。本论文的主要创新性工作包含以下几方面： 1.制备了具有强烈细胞黏附反差的微图案化表面的高分子水凝胶,并提出了适于细胞形状研究的黏附岛面积的判据以及基于显微照片统计干细胞分化程度的半定量方法。论文工作过程中制备了聚乙二醇(PEG)水凝胶表面精氨酸—甘氨酸—天冬氨酸(RGD)微图案,并借助于该高分子强烈抗拒细胞黏附的特征实现了细胞在黏附岛上的良好而持久的定位。在此基础上,建立了利用图案化表面的细胞显微照片判断骨髓基质干细胞成骨分化和成脂分化的统计方法,并且提出了最适于考察细胞形状效应的细胞可黏附的微岛面积的判据。这样,为成功实现不同形状和长径比的单细胞的定位,并合理、有效地基于微图案化表面研究单个或数个干细胞向成骨和成脂方向分化奠定了方法学的基础。 2.揭示细胞形状影响干细胞分化,并发现对于骨髓基质干细胞分化存在最佳长径比。利用我们课题组发展的PEG水凝胶表面微图案化技术,实现了骨髓基质干细胞的长时间定位,并在合适条件下持久维持所设定的圆形、方形、三角形、星形以及不同长径比的矩形等一系列不同形状,揭示细胞形状显著影响干细胞在成骨/成脂分化液中的分化程度,如：圆形细胞更易于向成脂肪细胞分化、星形细胞更易于向成骨细胞分化；并且在考察长径比效应时发现,虽然成脂分化随长径比增加单调下降,但是成骨分化随着长径比上升呈现峰值,即,存在最佳长径比。 3.系统研究了细胞尺寸、细胞接触和细胞密度对于干细胞分化的影响,利用化学图案化技术解构了细胞密度影响干细胞分化的内在物理因素。论文利用了微图案化技术,一种细胞接种密度下在不同黏附岛上实现了不同的局部密度,并且可以独立研究细胞尺寸、细胞之间的接触以及细胞密度的影响。结论证实,细胞尺寸和接触都会影响骨髓基质干细胞的成骨分化和成脂分化,而密度效应则是尺寸效应和接触效应的综合结果。随着细胞密度上升,对于成脂分化,细胞尺寸效应和接触效应给出同样的趋势；而对于成骨分化,细胞尺寸效应和细胞接触效应在细胞密度上升时则给出了“竞争”性的效应。我们在利用图案化技术解构细胞密度对干细胞分化影响的内在的物理因素的基础上,进一步提出了影响干细胞分化的细胞密度的两个区段的划分概念。
[Abstract]:The interaction of cells and materials is a basic scientific problem in the study of biological materials, cell biology and regenerative medicine. The relevant research is of great significance to the design of biological materials. The cell, as the basic unit of life activity, can directly reflect the changes of the intrinsic nature of the cell. However, there has been insufficient progress in this regard due to the relative shortage of efficient single-cell research instruments. the micro-pattern technology can realize the cell adhesion contrast on the substrate, so that the positioning of the cells can be controlled, the adhesion of the single cell can be effectively realized through the size of the cell adhesion area, that is, the size of the adhesion island, and the number of the samples can be used for counting and obtaining the credible conclusion, and has good application prospect in the research of single-cell. The tissue is not composed of single cells, but is one of several or more cells and a matrix of extracellular matrix. The interaction between the cells and the cells is also an important basic science in the tissue engineering Problem. The degree of aggregation of multicellular, that is, the cell density is also very high It is important that by controlling the positioning of the cells, the contact of the cells with the cells, the degree of aggregation of the cells, and the like can be effectively controlled by controlling the positioning of the cells, so that the interaction between the multiple cells can be effectively controlled. The bone marrow stromal stem cell has multi-directional differentiation potential and is easy to be amplified in vitro, and the bone marrow stromal stem cells are self-contained and not related to the field of ethics, and are ideal for tissue engineering. Subcell type. Stem cell differentiation is today's white-and-white science This dissertation uses bone marrow stromal stem cells as a model to study the various physical factors that affect the differentiation of stem cells, including the shape effect of single cell differentiation, the size effect, and the dry matter in the multi-cell state. The density effect of cell differentiation. The new work includes the following aspects: 1. Preparation of a strong cell adhesion contrast The micro-patterned surface of the micro-patterned surface is a high-molecular hydrogel, and a criterion for the area of the adhesion island suitable for cell shape study and a microphotograph system are proposed. A semi-quantitative method for determining the degree of differentiation of stem cells was prepared. The micro-pattern of arginine-glycinate (RGD) on the surface of water-gel of polyethylene glycol (PEG) was prepared in the working process, and the cell adhesion was strongly resisted by the polymer. On this basis, a statistical method for the determination of bone differentiation and adipocyte differentiation of bone marrow stromal stem cells was established by using the cell micrographs of the patterned surface, and the most suitable for investigating the shape of the cells was proposed. so as to successfully realize the positioning of single cells with different shapes and length-to-diameter ratios, and reasonably and effectively study the single or several stem cells into the bone and the fibroblast on the basis of the micropatterned surface. the foundation of the methodology was laid down in the direction of the fat, 2. The cell shape was revealed to affect the differentiation of the stem cells and was found to be The best aspect ratio exists in the differentiation of the bone marrow stromal stem cells. The long-time positioning of the bone marrow stromal stem cells is achieved by using the PEG hydrogel surface micropatterning technology developed by our research group, and the set circular, square, triangular, star shape is maintained for a long time under appropriate conditions. and a series of different shapes, such as rectangles of different length-to-length ratios, reveal that the shape of the cells significantly affects the degree of differentiation of the stem cells in the bone/ fat-forming differentiation liquid, such as, the circular cells are more easily differentiated into the fat cells, and the star-shaped cells are more likely to be fine to the bone Cell differentiation; and in the study of the length-to-length ratio, it was found that, although the adipocyte differentiation increased monotonically with the aspect ratio, it was found that the differentiation of the bone along with the length-length ratio The effect of cell size, cell contact and cell density on the differentiation of stem cells was studied by using the chemical patterning technique. The internal physical factors that influence the differentiation of the stem cells by the density of the cells are constructed. The paper makes use of the micropatterning technique, and a different local density is realized on the different adhesion islands under the density of cell inoculation, and the fine pattern can be studied independently. The effects of cell size, cell size, and cell density have been found to affect the differentiation and differentiation of bone marrow stromal stem cells into bone marrow stromal stem cells. The density effect is a comprehensive result of the size effect and the contact effect. As the cell density increases, the same trend is given for adipocyte differentiation, cell size effect and contact effect. The effect of "" on stem cell differentiation is further put forward based on the intrinsic physical factors of the influence of the density of the cells on the differentiation of the stem cells by using the patterning technique.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

【共引文献】